The present invention relates to chemical compounds having inhibitory activity on an intermediate conductance Ca2+ activated potassium channel (IKCa), and the use of such compounds for the treatment or alleviation of diseases or conditions relating to immune dysfunction.
Moreover, the invention relates to a method of screening a chemical compound for inhibitory activity on an intermediate conductance Ca2+ activated potassium channel (IKCa).
Ion channels are transmembrane proteins, which catalyse the transport of inorganic ions across cell membranes. The ion channels participate in processes as diverse as the generation and timing of action potentials, synaptic transmissions, secretion of hormones, contraction of muscles, etc.
Many drugs exert their effects via modulation of ion channels. Examples are anti-epileptic compounds like Phenytoin and Lamotrigine, which block voltage dependent Na+-channels in the brain, anti-hypertensive drugs like Nifedipine and Diltiazem, which block voltage dependent Ca2+-channels in smooth muscle cells, and stimulators of insulin release like Glibenclamide and Tolbutamide, which block an ATP-regulated K+-channel in the pancreas.
There is a large and still growing demand for non-toxic immunoregulating agents for use in relation to e.g. organ transplantation and auto-immune diseases.
Some of the currently used immunosuppressive compounds such as Cyclosporin A and FK506 prevent immunological proliferation by inhibition of the Ca2+/calmodulin-dependent Ser/Thr phosphatase calcineurin. The usefulness of this class of compounds is limited by their side effects such as renal dysfunction, arterial hypertension, neurological effects (headache, insomnia, tremors, parasthesias, lethargy), gastrointestinal effects (nausea, vomiting, diarrhoea), and diabetes.
Another class of compounds comprising e.g. Azathioprine and Mizorbine interfere in a cytotoxic manner directly with the DNA-replication process. Although cytotoxicity shows some selectivity towards strongly proliferating cells such as activated T- and B-lymphocytes, complications may follow due to effects on dividing cells in the entire body, including bone marrow, hair sacs, the skin, testis, ovary and epithelia such as the airways, the intestinal tract, and the thick ascending limp of the loop of Henle""s.
A fairly new approach for suppression of immune responses is to interfere with ion channels in the plasma membrane of cells in the immune system, especially the T- and B-lymphocytes. Upon exposure to antigens by antigen presenting macrophages or to mitogens such as IL-2 or IFN-xcex3, an initial signal in the switching from the resting phase to the proliferating phase is an activation of the phosphoinositide signalling pathway resulting in an increase in the intracellular concentration of Ca2+ ([Ca2+]i) due to Ca2+ release from intracellular stores. A sustained elevated [Ca2+])i is maintained by an increased passive influx through mitogen regulated, voltage-independent Ca-channels. This increase in [Ca2+]i is vital for the subsequent events leading to cell proliferation and secretion of lymphokines.
In resting T- and B-lymphocytes, the [Ca2+] is approximately 107 fold higher outside versus inside the cell, and the membrane potential is negative inside, i.e. there is an inwardly directed electrochemical Ca2+ gradient. Thus, when the Ca-channels are activated they conduct Ca2+ into the cell. However, Ca2+ influx via the Ca-channels, tends to reduce or even eliminate this gradient, and thus to reduce the influx. Concomitant opening of K-channels keeps the membrane potential negative, and activation of these channels is therefore essential for maintaining a large inwardly directed, electrochemical driving force for Ca2+.
In the presence of blockers of lymphocyte K-channels, the cells depolarise, and thereby the Ca2+ influx necessary for the activation of the immune response is reduced.
Several types of K-channels have been described in B- and T-lymphocytes including both voltage-dependent K-channels (Kv), and voltage-independent Ca2+-activated K-channels (KCa). It is well established, that the Kv-channels are activated by the Ca2+-induced depolarisation of the lymphocyte, and non-selective blockers of Kv-channels are therefore quite effective immunosuppressive agents. However, these compounds in general have severe side effects due to block of repolarisation in excitable tissue (seizures, myotonic runs, high blood pressure, etc.).
Considerable effort has been made into the development of immunoselective Kv-blockers. The molecular rationale for this, has been the observation that T-lymphocytes express homomeric Kv1.3-channels in contrast to excitable cells, which always express several heteromeric subtypes of the Kv-channels.
A selective blocker of the Kv1.3-homomer might therefore be an ideal, relatively non-toxic, immunosuppressive agent. Initial reports from these pharmacological programs indicate that selective Kv1.3-blockers are very effective as anti-inflammatory agents. However, the well-known toxicity of non-selective Kv-blockers has apparently not disappeared. An example is the potent Kv1.3 blocker CP-339,818. This compound is also a potent blocker of Kv1.4, a cardiac and neuronal A-type K-channel. The side-effect of this compound is predicted to be interference with the cardiac action potential (long QT-syndrome toxicity) as well as with the action potential repolarisation and after hyperpolarization in neurons.
A hitherto untested alternative to the block of the voltage-dependent K-channels is a selective inhibition of the Ca2+-activated K-channels in T- and B- lymphocytes. These channels are directly activated by the increased [Ca2+]i which is the primary signal for lymphocyte activation. Further, contrary to Kv-channels, these channels are voltage-independent, and therefore they do not close upon hyperpolarization, implicating that they are even more effective than Kv channels in maintaining a large inward driving force on Ca2+ under conditions of elevated intercellular Ca2+-concentrations.
Two types of Ca2+-activated K-channels have been described from lymphocytes: 1) Small-conductance, apamin-sensitive, Ca2+-activated K-channels (SKCa) and 2)xe2x88x92 Intermediate-conductance, inwardly rectifying, Clotrimazole-sensitive, Ca2+-activated K-channels (IKCa), also referred to as Gardos-channels. Resting T-lymphocytes express both SKCa and IKCa, whereas B-lymphocytes only express IKCa.
Upon activation, prior to cell proliferation, the expression level of IKCa increases approximately 30 fold in both T- and B-lymphocytes. The expression levels of both Kv1.3 and SKCa remain unchanged, indicating a major role for the IKCa-channel in induction of T- and B-cell proliferation. Contrary to the SKCa-channels, which are extensively expressed in CNS and heart (measured as mRNA abundance by Northern hybridisation) and in PNS, skeletal muscle, hepatocytes (measured as functional channels by electrophysiology), expression of IKCa-channels have never been reported from any excitable tissue. In fact, blood cells such as erythrocytes, monocytes, lymphocytes, endothelial cells, and certain cell-lines with an epithelial ancestry, Ehrlich ascites tumor cells and HeLa cells appear to be the main source of this type of channels.
Furthermore, the very recent cloning of IKCa has enabled the demonstration of the mRNA for this gene in several organs including placenta, salivary glands, lung and pancreas. Thus, specific blockers of IKCa are likely to be very effective as immunosuppressive agents, and devoid of side effects on excitable tissue. In fact, the IKCa-inhibitor Clotrimazole (which is also a blocker of the cytochrome P-450 system) has been extensively used clinically in the systemic treatment of fungal infections. No toxicity related to K-channel blockade has been described.
Accordingly, in its first aspect, the invention relates to the use of a chemical compound having IKCa inhibitory activity for the manufacture of a medicament for the treatment or alleviation of diseases, disorders or. conditions relating to immune dysfunction.
In another aspect the invention provides a pharmaceutical compositions for use in the treatment or alleviation of diseases, disorders or conditions relating to immune dysfunction, comprising an effective amount of a chemical compound having IKCa inhibitory activity.
In yet another aspect the invention provides a method of screening a chemical compound for inhibitory activity on an intermediate conductance Ca2+ activated potassium channel (IKCa), which method comprises the steps of subjecting an IKCa containing cell to the action of the chemical compound, and monitoring the membrane potential of the IKCa containing cell.
The present invention relates to the use of a chemical compound having IKCa inhibitory activity for treatment or alleviation of diseases or conditions relating to immune dysfunction.
Chemical Compound having IKCa Inhibitory Activity
According to the invention, chemical compound having IKCa inhibitory activity may be identified by its ability to inhibit hyperpolarization of an IKCa containing cell, i.e. a cell containing an intermediate conductance Ca2+activated potassium channel (IKCa). In a preferred embodiment, the chemical compounds having IKCa inhibitory activity is identified by the method of screening described below.
Preferred chemical compounds having IKCa inhibitory activity for use according to the invention are the derivatives of 1,4-dihydropyridine-3,5-dicarboxylic acid, the imidazole derivatives, the triazole derivatives, the nitroimidazole derivatives, and the derivatives and metabolites of Clotrimazole, described below. The derivatives of 1,4-dihydropyridine-3,5-dicarboxylic acid have been disclosed in e.g. U.S. Pat. No. 3,799,934. The imidazole derivatives, the triazole derivatives, and the nitroimidazole derivatives have been disclosed in e.g. U.S. Pat. No. 5,273,992. The derivatives and metabolites of Clotrimazole have been disclosed in e.g. WO 96/08242.
Derivatives of 1,4-dihydropyridine-3,5-dicarboxylic acid
In a preferred embodiment, the chemical compound having IKCa inhibitory activity for use according to the invention is a symmetric or asymmetric derivative of 1,4-dihydropyridine-3,5-dicarboxylic acid represented by the general formula 
wherein
R represents an alkyl group or a cycloalkyl group;
or R represents a mono- or polycyclic aryl group, which aryl group may be substituted one or more times with substituents selected from the grgroup consisting of halogen, trifluoromethyl (xe2x80x94CF3), nitro (xe2x80x94NO2), cyano (xe2x80x94CN), azido (xe2x80x94N3), a group of the formula xe2x80x94S(O)n-alkyl, xe2x80x94S(O)nxe2x80x94NH-alkyl, or xe2x80x94S(O)nxe2x80x94N-(alkyl)2, in which n has a value of 0, 1 or 2, an alkyl group, a cycloalkyl group, an alkoxy group, a trifluoromethyl-oxy group (xe2x80x94OCF3), a carboxy group (xe2x80x94COOH), a group of the formula xe2x80x94COO-alkyl, a carbamoyl group (xe2x80x94CONH2), and a group of the formula xe2x80x94CONH-alkyl or xe2x80x94CON(alkyl)2;
or R represents a mono- or poly-heterocyclic group, which heterocyclic group may be substituted one or more times with alkyl, alkoxy, a carboxy group (xe2x80x94COOH), a group of the formula xe2x80x94COO-alkyl, and/or a group of the formula xe2x80x94COO-phenyl;
and R1, R2, R3 and R4, independent of each another, represents hydrogen, an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an alkoxy group, a phenyl group, a phenyl-alkyl group, a furanyl group, a furanyl-alkyl group, a pyridyl group, or a pyridyl-alkyl group;
or a pharmaceutically acceptable acid addition salt thereof.
In a more preferred embodiment, the chemical compound for use according to the invention is a compound of the general formula (I) in which R represents a cyclohexyl group; or R represents a monosubstituted phenyl group, which phenyl group may be substituted one or more times with substituents selected from the group consisting of halogen, trifluoromethyl (xe2x80x94CF3), nitro (xe2x80x94NO2), and cyano (xe2x80x94CN); or R represents a pyridyl group or a dihydro-pyridyl group, which groups may be monosubstituted with a group of the formula xe2x80x94COO-alkyl, or a group of the formulaxe2x80x94COO-phenyl.
In a another preferred embodiment, the chemical compound for use according to the invention is a compound of the general formula (I) in which R represents a 2-nitrophenyl group, a 3-nitrophenyl group, a 4-nitrophenyl group, a 2-trifruoromethylphenyl group, a 3-trifruoromethylphenyl group, or a 4-trifruoromethylphenyl group; a 2-cyanophenyl group, a 3-cyanophenyl group, a 4-cyanophenyl group; or R represents a 2-pyridyl, a 3-pyridyl or a 4-pyridyl group, a 1,2-, 1,4- or 1,6-dihydro-2-pyridyl, a 1,2-, 1,4- or 1,6-dihydro-3-pyridyl, or a 1,2- or 1,4-dihydro-4-pyridyl group, which pyridyl or dihydropyridyl groups may be monosubstituted with C1-6-alkyl, a group of the formula xe2x80x94COOxe2x80x94C1-6-alkyl, or a group of the formula xe2x80x94COO-phenyl.
In yet another preferred embodiment, the chemical compound for use according to the invention is a compound of the general formula (I) in which R1, R2, R3 and R4, independent of each another, represents C1-6-alkyl, preferably methyl, ethyl, propyl, isopropyl, butyl, or isobutyl.
In a more preferred embodiment, the chemical compound for use according to the invention is a compound of the general formula (I) which is an asymmetric derivative of the 1,4-dihydropyridine-3,5-dicarboxylic acid represented by the general formula (I). Preferred asymmetric derivatives includes asymmetric C1-6-alkyl derivatives of the 1,4-dihydropyridine-3,5-dicarboxylic acid represented by the general formula (I). Most preferred asymmetric compounds include
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester (Nitrendipine);
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-nitrophenyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-nitrophenyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-nitrophenyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-trifluorophenyl)pyrdine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-trifluorophenyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-trifluorophenyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-trifluorophenyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-trifluorophenyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-trifluorophenyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-trifluorophenyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-trifluorophenyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-trifluorophenyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-cyanophenyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-cyanophenyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-cyanophenyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-cyanophenyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-cyanophenyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-cyanophenyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-cyanophenyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-cyanophenyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-cyanophenyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-pyridyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-pyridyl)pyridine-3,5-dicarboxylic acid propyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-pyridyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-pyridyl)pyridine-3,5-dicarboxylic acid ethyl methyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-pyridyl)pyridine-3,5-dicarboxylic acid propyl methyl ester; and
1,4-dihydro-2,6-dimethyl-4-(4-pyridyl)pyridine-3,5-dicarboxylic acid isopropyl methyl ester.
In another preferred embodiment, the chemical compound is a symmetric derivative of 1,4-dihydropyridine-3,5-dicarboxylic acid represented by the general formula (I). Preferred chemical compounds include the symmetric C1-6-alkyl derivatives of the 1,4-dihydropyridine-3,5-dicarboxylic acid. Most preferred symmetric chemical compounds for use-according to the invention include
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid dimethyl ester (Nifedipine);
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-nitrophenyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-nitrophenyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-trifluorophenyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-trifluorophenyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-trifluorophenyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-trifluorophenyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-trifluorophenyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-trifluorophenyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-cyanophenyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-cyanophenyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-cyanophenyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-cyanophenyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-cyanophenyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-cyanophenyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-pyridyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(3-pyridyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-pyridyl)pyridine-3,5-dicarboxylic acid dimethyl ester;
1,4-dihydro-2,6-dimethyl-4-(4-pyridyl)pyridine-3,5-dicarboxylic acid diethyl ester;
1,4-dihydro-2,6-dimethyl-4-cyclohexylpyridine-3,5-dicarboxylic acid dimethyl ester; and
1,4-dihydro-2,6-dimethyl-4-cyclohexylpyridine-3,5-dicarboxylic acid diethyl ester.
Imidazole Derivatives
In another preferred embodiment, the chemical compound having IKCa inhibitory activity for use according to the invention is an imidazole derivative selected from the group consisting of
1-[(2-chlorophenyl)-diphenyl-methyl]-1H-imidazole (Clotrimazole);
1-[(3-chlorophenyl)-diphenyl-methyl]-1H-imidazole;
1-[(4-chlorophenyl)-diphenyl-methyl]-1H-imidazole;
1-[(2-chlorophenyl)-(4-hydroxyphenyl)-phenyl-methyl]-1H-imidazole;
1-[(3-chlorophenyl)-(4-hydroxyphenyl)-phenyl-methyl]-1H-imidazole;
1-[(4-chlorophenyl)-(4-hydroxyphenyl)-phenyl-methyl]-1H-imidazole;
1-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy]-1H-imidazole (Miconazole);
1-Acetyl-4[4-[(2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4yl]methoxy]phenyl]piperazine (Ketoconazole);
1-[2-[(4-chlorophenyl)methoxyl]-2-(2,4-dichlorophenyl)ethyl]-1H-imidacole (Econazole);
1-[4-(4-chlorophenyl)-2-(2,6-dichlorophenylthio)butyl]imidazole mononitrate (Butoconazole);
2xe2x80x2,4xe2x80x2-dichloro-2-imidazol-1-ylacetophenone-(Z)-O-(2,4-dichlorobenzyl)oxime mononitrate (Oxiconazole);
1-[2,4-dichloro-xcex2-(4-chlorobenzyl)thiophenethyl]imidazole nitrate (Sulconazole); and
1-[2-[(2-chloro-3-thienyl)methoxy]-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole (Thioconazole).
Triazole Derivatives
In a third preferred embodiment, the chemical compound having IKCa inhibitory activity for use according to the invention is a triazole derivative selected from the group consisting of
2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol (Fluconazole);
1-{4-[[2-(2,4-dichlorophenyl)r-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-c-4-yl]methoxy]-phenyl}-4-isopropylpiperazine (Terconazole);
(xc2x1)-2-sec-butyl-4-[4-(4-{4-[(2R*,4S*)-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1ylmethyl)-1,3-dioxolan-4-ylmethoxy]phenyl}-piperazin-1-yl)phenyl]-2,4-dihydro-1,2,4-triazol-3-one (Itraconazole).
Nitroimidazole Derivatives
In a fourth preferred embodiment, the chemical compound having IKCa inhibitory activity for use according to the invention is a nitroimidazole derivative selected from the group consisting of
2-methyl-5-nitroimidazole-1-ethanol (Metronidazole);
1-[2-(ethylsulphonyl)ethyl]-2-methyl-5-nitroimidazole (Tinidazole);
4-[2-(5-nitroimidazol-1-yl)ethyl]morpholine (Nimorazole);
1-chloro-3-(2-methyl-5-nitroimidazol-1-yl)propan-2-ol (Omidazole), and
N-benzyl-2-(2-nitroimidazol-1-yl)acetamide (Benznidazole).
Metabolites of Clotrimazole
In yet another preferred embodiment chemical compounds having IKCa inhibitory activity for use according to the invention are derivatives and metabolites of Clotrimazole, as described in WO 96/08242.
The derivatives and metabolites of Clotrimazole for use according to the invention may be characterised by the following general formula 
wherein
X represents halogen, a trifluoromethyl group, a nitro group, or a cyano group;
R represents hydrogen, halogen, hydroxy, an alkyl group, a cycloalkyl group, an alkoxy group, or an alkyloxy group;
R1 represents hydrogen, or a phenyl group, which phenyl group may be substituted one or more times with substituents selected from the group consisting of halogen and hydroxy;
R2 represents hydrogen, hydroxyl, alkyl, alkoxy;
R3 represents a group of the formula xe2x80x94Yxe2x80x94CH2xe2x80x94R5, wherein Y represents oxygen (xe2x80x94Oxe2x80x94) or sulphur (xe2x80x94Sxe2x80x94); a group of the formula xe2x95x90NOxe2x80x94CH2R5; a group of the formula xe2x80x94O-phenylxe2x80x94CHxe2x95x90CH2; a group of the formula xe2x80x94CH2xe2x80x94CH(CH3)xe2x80x94S-phenyl, which phenyl may be substituted one or more times with substituents selected from the group consisting of halogen and hydroxy; or a phenyl group, which phenyl may be substituted one or more times with substituents selected from the group consisting of halogen and hydroxy; and wherein R5 represents an ethenyl group (CH2xe2x95x90CHxe2x80x94); a phenyl group, which phenyl may be substituted one or more times with substituents selected from the group consisting of halogen and hydroxy; a phenyl-S-phenyl group, a group of the formula CH2xe2x80x94O-phenyl, which phenyl may be substituted one or more times with substituents selected from the group consisting of halogen and hydroxy; or a group of the formula 
xe2x80x83wherein Z represents S, O or N;
and R6 represents hydrogen, halogen or hydroxy;
or a pharmaceutically acceptable acid addition salt thereof.
Preferred derivatives and metabolites for use according to the invention include
2-chlorophenyl-4-hydroxyphenyl-phenyl-methane;
2-chlorophenyl-bis-phenyl-methane;
2-chlorophenyl-bis-phenyl-methanol;
3-(1-[2,4-dichlorophenyl]-ethoxymethyl)-2-chlorothiophene;
0-(2,4-dichlorobenzyl)-2,4-dichloroacetophenone oxime;
1-(2,4-dichloro)-1-(4-(phenylthio)benzyloxy)ethane;
1-(2,4-dichlorophenyl) 1-1(allyloxy)ethane;
1-(2,4-dichlorophenyl)-1-(4-chlorobenzylthio)ethane;
1-(2,4-dichlorophenyl)-1-(2,4-dichlorobenzyloxy)ethane;
1-(2,4-dichlorophenyl)ethyl-2,6-dichlorobenzyl ether;
1-(2-[4-chlorophenoxy]ethyloxy)-1-(2,4-dichlorophenyl)propene;
1-(2,4-dichlorophenyl)-ethyl-(4-chlorophenyl)methyl ether;
3-chlorobenzyl-2-vinylphenyl ether; and
1-(4-chlorophenyl)-3-(2,6-dichlorophenylthio)butane.
Definition of Substituents
In the context of this invention halogen represents a fluorine, a chlorine, a bromine or a iodine atom.
In the context of this invention an alkyl group designates a univalent saturated, straight or branched hydrocarbon chain. The hydrocarbon chain preferably contain of from one to eighteen carbon atoms (C1-8-alkyl), more preferred of from one to six carbon atoms (C1-6-alkyl; lower alkyl), including pentyl, isopentyl, neopentyl, tertiary pentyl, hexyl and isohexyl. In a preferred embodiment alkyl represents a C1-4-alkyl group, including butyl, isobutyl, secondary butyl, and tertiary butyl. In a most preferred embodiment alkyl represents a C1-3-alkyl group, which may in particular be methyl, ethyl, propyl or isopropyl.
In the context of this invention a cycloalkyl group designates a cyclic alkyl group, preferably containing of from three to seven carbon atoms (C3-7-cycloalkyl), including cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
In the context of this invention an alkenyl group designates a carbon chain containing one or more double bonds, including di-enes, tri-enes and poly-enes. In a preferred embodiment the alkenyl group of the invention comprises of from two to six carbon atoms (C2-6-alkenyl), including at least one double bond. In a most preferred embodiment the alkenyl group of the invention is ethenyl; 1,2- or 2,3-propenyl; or 1,2-, 2,3-, or 3,4-butenyl.
In the context of this invention an alkynyl group designates a carbon chain containing one or more triple bonds, including di-ynes, tri-ynes and poly-ynes. In a preferred embodiment the alkynyl group of the invention comprises of from two to six carbon atoms (C2-6-alkynyl), including at least one triple bond. In its most preferred embodiment the alkynyl group of the invention is ethynyl, 1,2- or 2,3-propynyl, 1,2-, 2,3- or 3,4-butynyl.
In the context of this invention an alkoxy group designates an xe2x80x9calkyl-Oxe2x80x94xe2x80x9d group, wherein alkyl is as defined above.
In the context of this invention a mono- or polycyclic aryl group designates a monocyclic or polycyclic aromatic hydrocarbon group. Examples of preferred aryl groups of the invention are phenyl, naphthyl and anthracenyl.
In the context of this invention a mono- or poly-heterocyclic group is a mono- or polycyclic aromatic group, which holds one or more heteroatoms in its ring structure. Preferred heterocyclic monocyclic groups of the invention are 5- and 6 membered heterocyclic monocyclic groups. Examples of preferred heterocyclic monocyclic groups of the invention include furanyl, imidazolyl, isoimidazolyl, 2-isoimidazolyl, isothiazolyl, isoxazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, and thienyl. Examples of preferred heterocyclic polycyclic groups of the invention include benzimidazolyl, indolyl, isoquinolyl and quinolyl.
The chemical compounds for use according to the invention have been described and may be prepared by methods known in the art.
Pharmaceutically Acceptable Salts
The chemical compound of the invention may be provided in any form suitable for the intended administration. Suitable forms include pharmaceutically (i.e. physiologically) acceptable salts, or pre- or prodrug forms of the chemical compound of the invention.
Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzenesulfonate derived from benzensulfonic acid, the benzoate derived from benzoic acid, the cinnamate derived from cinnamic acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the formate derived from formic acid, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the hydrochloride derived from hydrochloric acid, the hydrobromide derived from hydrobromic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the malonate derived from malonic acid, the mandelate derived from mandelic acid, the methanesulfonate derived from methane sulphonic acid, the naphthalene-2-sulphonate derived from naphtalene-2-sulphonic acid, the nitrate derived from nitric acid, the perchlorate derived from perchloric acid, the phosphate derived from phosphoric acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acids the sulphate derived from sulphuric acid, the tartrate derived from tartaric acid, the toluene-p-sulphonate derived from p-toluene sulfonic acid, and the like. Such salts may be formed by procedures well known and described in the art.
Other acids such as oxalic acid, which may not be considered pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining a chemical compound of the invention and its pharmaceutically acceptable acid addition salt.
Metal salts of a chemical compound of the invention includes alkali metal salts, such as the sodium salt, of a chemical compound of the invention containing a carboxy group.
The chemical compound of the invention may be provided in dissoluble or indissoluble forms together with a pharmaceutically acceptable solvents such as water, ethanol, and the like. Dissoluble forms may also include hydrated forms such as the monohydrate, the dihydrate, the hemihydrate, the trihydrate, the tetrahydrate, and the like. In general, the dissoluble forms are considered equivalent to indissoluble forms for the purposes of this invention.
Steric Isomers
The chemical compounds of the present invention may exist in (+) and (xe2x88x92) forms as well as in racemic forms. The racemates of these isomers and the individual isomers themselves are within the scope of the present invention.
Racemic forms can be resolved into the optical antipodes by known methods and techniques. One way of separating the diastereomeric salts is by use of an optically active acid, and liberating the optically active amine compound by treatment with a base. Another method for resolving racemates into the optical antipodes is based upon chromatography on an optical active matrix. Racemic compounds of the present invention can thus be resolved into their optical antipodes, e.g., by fractional crystallisation of d- or I- (tartrates, mandelates, or camphorsulphonate) salts for example.
The chemical compounds of the present invention may also be resolved by the formation of diastereomeric amides by reaction of the chemical compounds of the present invention with an optically active activated carboxylic acid such as that derived from (+) or (xe2x88x92) phenylalanine, (+) or (xe2x88x92) phenylglycine, (+) or (xe2x88x92) camphanic acid or by the formation of diastereomeric carbamates by reaction of the chemical compound of the present invention with an optically active chloroformate or the like.
Additional methods for the resolving the optical isomers are known in the art. Such methods include those described by Jaques J, Collet A, and Wilen S in xe2x80x9cEnantiomers, Racemates, and Resolutionsxe2x80x9d, John Wiley and Sons, New York (1981).
Moreover, some of the chemical compounds of the invention being oximes, may thus exist in two forms, syn- and anti-form (Z- and E-form), depending on the arrangement of the substituents around the xe2x80x94Cxe2x95x90Nxe2x80x94 double bond. A chemical compound of the present invention may thus be the syn- or the anti-form (Z- and E-form), or it may be a mixture hereof.
Method of Screening
In another aspect, the present invention provides a method for the screening of chemical compounds for inhibitory activity on an intermediate conductance Ca2+ activated potassium channel (IKCa), by which method a chemical compound having IKCa inhibitory activity is identified by its ability to inhibit hyperpolarization of the cell.
The screening method of the invention comprises the steps of
subjecting an IKCa containing cell to the action of the chemical compound to be screened, and
monitoring the membrane potential of the IKCa containing cell.
More particularly the monitoring of the membrane potential of the IKCa containing cell of step (ii) is carried out in order to monitor changes in the membrane potential caused by the action of the chemical compound.
The IKCa Containing Cell
The IKCa used in the method of the invention may be of any origin, however, preferably of human or animal origin. Also, the IKCa may be endogenous or it may be exogenous to the cell in question.
In a preferred embodiment, the IKCa of the IKCa containing cell is an ion channel that is endogenous to the cell in question, and which cell may in particular be a T- or B-lymphocyte or other cells known to express IKCa, e.g. a HeLa cell, or a cell of epithelial origin, a cell of endothelial origin, or a blood cell.
In another preferred embodiment, the IKCa of the IKCa containing cell is an ion channel that is exogenous to the cell in question, and which cell may in particular be a human embryonic kidney (HEK) cell, a HEK 293 cell, a Chinese hamster ovary (CHO) cell, a Xenopus laevis oocyte (XLO) cell, or any other cell line able to express IKCa.
The IKCa preferably is of human origin. In particular the IKCa may be isolated from salivary glands, from lung tissue, from tracheal tissue, from placenta tissue, from pancreas tissue, from lymphocytes, from colon tissue, from kidney tissue, from thymus tissue, from bone marrow, from prostate tissue, from stomach tissue, from liver tissue, from foetal liver tissue, from mammary glands, from small intestine tissue, from spleen tissue, or from lymph node tissue. Preferably the IKCa may be isolated from salivary glands, from lung tissue, from tracheal tissue, from placenta tissue, from pancreas tissue, or from lymphocytes.
In a most preferred embodiment, the IKCa is encoded by the DNA sequence presented as SEQ ID NO: 1, or a homologous sequence, e.g. a DNA sequence showing a homology to SEQ ID NO: 1 of at least 80%, more preferred at least 90%, most preferred at least 95%.
Monitoring of the Membrane Potential
According to the method of the invention the membrane potential is monitored in order to determine changes in the membrane potential. The membrane potential may be monitored using established methods.
In a preferred embodiment monitoring of the membrane potential of the IKCa containing cell is performed by patch clamp techniques, e.g. as described by Hamill, O. P., et al., Pflxc3xcgers Arch. 1981 351 85-100. In a more preferred embodiment, monitoring of the membrane potential of the IKCa containing cell is performed by the automatic patch clamp method described in pending patent application DK 1151/97.
In another preferred embodiment monitoring of the membrane potential of the IKCa containing cell is performed using fluorescence methods.
In a preferred method of the invention, the IKCa containing cell is mixed with a membrane potential indicating agent, that allow for a determination of changes in the membrane potential of the cell, caused by the addition of the test compound.
The membrane potential indicating agent employed in the method of the invention may be any agent that allow monitoring of changes in the membrane potential. In a preferred embodiment, the membrane potential indicating agent is a fluorescent indicator. The fluorescent indicator must be sufficiently sensitive so as to produce a detectable change in fluorescence intensity in the presence of calcium ions.
Preferred fluorescent indicators are in particular DIBAC4(3), DiOC5(3), and DIOC2(3).
Monitoring of the membrane potential of the IKCa containing cell may then be performed by spectroscopic methods, e.g. using a FLIPR assay (Fluorescence Image Plate Reader; available from Molecular Devices), or by using the automated analysis equipment described in U.S, Pat. No. 5,670,113.
In a separate aspect the invention relates to an encompasses the chemical compounds identified by the method of the invention and their use the use of these compounds for the treatment or alleviation of diseases or conditions relating to immune dysfunction.
Biological Activity
As described above, the IKCa inhibitory compounds of the invention are useful as immune modulating agents, i.e. agents capable of regulating the immune system. More particularly, the IKCa inhibitory compounds of the present invention may be used for reducing or inhibiting undesired immunoregulatory actions.
In a preferred embodiment, the invention relates to the use of an IKCa inhibitory compound for the treatment or alleviation of a diseases, disorders or condition related to immune dysfunction.
Conditions which may benefit from this treatment include, but are not limited to diseases, disorders or conditions such as autoimmune diseases, e.g. Addison""s disease, alopecia areata, Ankylosing spondylitis, hemolytic anemia (anemia haemolytica), pernicious anemia (anemia perniciosa), aphthae, aphthous stomatitis, arthritis, osteoarthritis, rheumatoid arthritis, aspermiogenese, asthma bronchiale, autoimmune asthma, autoimmune hemolysis, Bechet""s disease, Boeck""s disease, inflammatory bowel disease, Burkitt""s lymphoma, Chron""s disease, chorioiditis, colitis ulcerosa, Coeliac disease, cryoglobulinemia, dermatitis herpetiformis, dermatomyositis, insulin-dependent type I diabetes, juvenile diabetes, idiopathic diabetes insipidus, insulin-dependent diabetes mellisis, autoimmune demyelinating diseases, Dupuytren""s contracture, encephalomyelitis, encephalomyelitis allergica, endophthalmia phacoanaphylactica, enteritis allergica, autoimmune enteropathy syndrome, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, glomerulo nephritis, Goodpasture""s syndrome, Graves"" disease, Hamman-Rich""s disease, Hashimoto""s disease, Hashimoto""s thyroiditis, sudden hearing loss, sensoneural hearing loss, hepatitis chronica, Hodgkin""s disease, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, iritis, leucopenia, leucemia, lupus erythematosus disseminatus, systemic lupus erythematosus, cutaneous lupus erythematosus, lymphogranuloma malignum, mononucleosis infectiosa, myasthenia gravis, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, pemphigus, pemphigus vulgaris, polyarteritis nodosa, polyarthritis chronica primaria, polymyositis, polyradiculitis acuta, psoreasis, purpura, pyoderma gangrenosum, Quervain""s hyreoiditis, Reiter""s syndrome, sarcoidosis, ataxic sclerosis, progressive systemic sclerosis, scleritis, sclerodermia, multiple sclerosis, sclerosis disseminata, acquired spenic atrophy, infertility due to antispermatozoan antobodies, thrombocytopenia, idiopathic thrombocytopenia purpura, thymoma, acute anterior uveitis, vitiligo, AIDS, HIV, SCID and Epstein Barr virus associated diseases such as Sjorgren""s syndrome, virus (AIDS or EBV) associated B cell lymphoma, parasitic diseases such as Lesihmania, and immunosuppressed disease states such as viral infections following allograft transplantations, graft vs. Host syndrome, transplant rejection, or AIDS, cancers, chronic active hepatitis diabetes, toxic chock syndrome, food poisoning, and transplant rejection.
Accordingly, in further embodiments, the invention relates to a chemical compound having IKCa inhibitory activity for use as a medicament.
More specifically the invention relates to the use of a chemical compound having IKCa inhibitory activity for use in the manufacture of a medicament for the treatment of treatment of diseases related to immune dysfunction. In a preferred embodiment the medicament is an immune system suppressing medicament (an immunosuppressivum).
Pharmaceutical Compositions
In yet another aspect the invention relates to pharmaceutical compositions for use in the treatment or alleviation of diseases, disorders or conditions related to immune dysfunction, which pharmaceutical composition comprises a therapeutically effective amount of a chemical compound having IKCa inhibitory activity, as identified by the method of the invention.
While a chemical compound of the invention for use in therapy may be administered in the form of the raw chemical compound, it is preferred to introduce the active ingredient, optionally in the form of a physiologically acceptable salt, in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries.
In a preferred embodiment, the invention provides pharmaceutical compositions comprising the chemical compound of the invention or a pharmaceutically acceptable salt or derivative thereof together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The pharmaceutical composition of the invention may be administered by any convenient route which suite the desired therapy. Preferred routes of administration include oral administration, in particular in tablet, in capsule, in dragxc3xa9, in powder, or in liquid form, and parenteral administration, in particular cutaneous, subcutaneous, intramuscular, or intravenous injection. The pharmaceutical composition may be prepared by the skilled person using standard and conventional techniques appropriate to the desired formulation. When desired, compositions adapted to give sustained release of the active ingredient may be employed.
The actual dosage depend on the nature and severity of the disease being treated, and is within the discretion of the physician, and may be varied by titration of the dosage to the particular circumstances of this invention to produce the desired therapeutic effect. However, it is presently contemplated that pharmaceutical compositions containing of from about 0.1 to about 500 mg of active ingredient per individual dose, preferably of from about 1 to about 100 mg, most preferred of from about 1 to about 10 mg, are suitable for therapeutic treatments.
The active ingredient may be administered in one or several doses per day. A satisfactory result can, in certain instances, be obtained at a dosage as low as 0.1 xcexcg/kg i.v. and 1 xcexcg/kg p.o. The upper limit of the dosage range is presently considered to be about 10 mg/kg i.v. and 100 mg/kg p.o. Preferred ranges are from about 0.1 xcexcg/kg to about 10 mg/kg i.v., and from about 1 xcexcg/kg to about 100 mg/kg p.o.
In a preferred embodiment, the pharmaceutical composition of the invention comprises a chemical compound which is an imidazole derivative, in particular Clotrimazole, Miconazole, Ketonazole, Econazole, Butoconazole, Oxiconazole, Sulconazole, or Tioconazole.
In another preferred embodiment, the pharmaceutical composition of the invention comprises a chemical compound which is a nitroimidazole derivative, in particular Metronidazole, Tinidazole, Nimorazole, Ornidazole, or Benznidazole.
In yet another preferred embodiment, the pharmaceutical composition of the invention comprises a chemical compound which is a triazole derivative, in particular Fluconazole, Tercolazole, or Itraconazole.
In a further preferred embodiment, the pharmaceutical composition of the invention comprises a chemical compound which is a metabolite of Clotrimazole, in particular 2-chlorophenyl-4-hydroxy-phenyl-phenyl-methane, 2-chlorophenyl-bis-phenyl-methane, or 2-chlorophenyl-bis-phenyl-methanol.
Method of Treatment
The IKCa inhibitory compounds of the invention are useful as immune modulating agents, i.e. agents capable of regulating the immune system, and may be used in a method of for reducing or inhibiting undesired immunoregulatory actions.
Therefore, in a separate aspect, the invention relates to a method of treatment or alleviation of diseases, disorders or conditions relating to immune dysfunction in a living body, said method comprising administering to said living body an effective amount of a chemical compound having IKCa inhibitory activity.